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brain research 1619 (2015) 8490

Available online at www.sciencedirect.com

www.elsevier.com/locate/brainres

Research Report

Myelomeningocele: How we can improve the


assessment of the most severe form of spina bida

George M. Smitha,b, Barbara Krynskaa,c,n


a
Shriners Hospitals Pediatric Research Center and Center for Neural Repair and Rehabilitation, Temple University School
of Medicine, Philadelphia, PA 19140, USA
b
Department of Neuroscience, Temple University School of Medicine Philadelphia, PA 19140, USA
c
Department of Neurology, Temple University School of Medicine Philadelphia, PA 19140, USA

ar t ic l e in f o abs tra ct

Article history: Myelomeningocele (MMC) is a devastating spinal cord birth defect, which results in
Accepted 25 November 2014 signicant life-long disabilities, impaired quality of life, and difcult medical management.
Available online 9 December 2014 The pathological progression of MMC involves failure in neural tube and vertebral arch

Keywords: closure at early gestational ages, followed by subsequent impairment in spinal cord and

Myelomeningocele vertebral growth during fetal development. MMC is irreversible at term. Thus, prenatal

Spina bida therapeutic strategies that interrupt progressive pathological processes offer an appealing

Birth defects approach for treatment of MMC. However, a thorough understanding of pathological

Spinal cord injury progression of MMC is mandatory for appropriate treatment to be rendered.

Vertebral defects This article is part of a Special Issue entitled SI: Spinal cord injury.

Micro-computed tomography & 2014 Elsevier B.V. All rights reserved.

1. Introduction in available imaging techniques can be used to precisely


identify and quantitatively evaluate the defect or repair status
In this review, we discuss the MMC spinal cord lesion with of MMC.
emphasis on the associated vertebral defects and how the use This review highlights the background and rationale for
of sensitive and quantitative outcome measures can precisely the use of 3D high resolution micro-computed tomography
identify and quantify changes in spinal cord and vertebral (micro-CT) imaging and digital analyses methods for
defects to better characterize progression and identify treat- improved quantitative assessment of MMC defects in animal
ments for MMC. The accurate identication and quantitative models. We propose that the inclusion of 3D imaging and
evaluation of MMC abnormalities is challenging because MMC quantitative measures of MMC defects using high-resolution
is a highly complex defect, characterized by a dynamic malfo- micro-CT will provide a deeper understanding of pathophy-
rmation process during gestation and varying degrees of siology or repair strategy of MMC in animal models, and
neural tissue loss and vertebral defects. Recent renements make the studies of MMC more effective and efcient.

n
Correspondence to: Shriners Hospitals Pediatric Research Center, Temple University School of Medicine, 3500 N Broad Street,
Philadelphia, PA 19140, USA. Fax: 1 215 707 8235.
E-mail address: krynskb@tuhs.temple.edu (B. Krynska).

http://dx.doi.org/10.1016/j.brainres.2014.11.053
0006-8993/& 2014 Elsevier B.V. All rights reserved.
brain research 1619 (2015) 8490 85

during the last decade, resulting in the two-hit hypothesis. The


2. MMC the problem rst hit, denotes the initial defect of neurulation and the
secondary hit represents the subsequent in utero acquired
Myelomeningocele (MMC), the most severe form of spina bida, damage to the openly exposed spinal cord (Meuli and
is a common and devastating congenital malformation and a Moehrlen, 2013). Although, failure during neurulation is the
major source of disabilities and morbidity in children. MMC initial cause for the spinal cord malformation, compelling
affects less than one of 1000 live births in the United States clinical as well as experimental evidence suggest that the
(Parker et al., 2010). About 75% of affected children survive to exposed spinal cord results in secondary in utero damage,
adulthood, but, live with signicant life-long physical disabil- predominantly causing the neurological decit associated with
ities (Bowman et al., 2001). The exact cause of spina bida is MMC (Heffez et al., 1990; Meuli et al., 1995b; Sival et al., 1997;
unknown; however, teratogenic, nutritional and genetic factors Stiefel et al., 2007). Human studies show that the openly exposed
are associated with the development of spina bida (Alles and spinal cord is histologically intact in early gestation, showing
Sulik, 1990; Kibar et al., 2007; Ornoy, 2009). Although certain risk little damage; however, progressive damage sometimes leading
factors are known to be associated with the development of to complete loss, was observed in late stages of gestation
spina bida, and the prevalence of spina bida has declined in (Hutchins et al., 1996; Meuli et al., 1997). In support of the in
the post-fortication era with the use of folic acid supplemen- utero acquired secondary damage, experimental evidence
tation to reproductive age women, spina bida still can affect demonstrated that surgically induced in utero exposure of the
the pregnancy of any women, at any age, or of any baseline spinal cord (human-like spina bida lesions) in experimental
health status (Moldenhauer, 2014; Osterhues et al., 2013). animals (rat, pig, sheep) led to spinal cord damage of the
Despite the clinical impact of MMC, there is no cure for this exposed segments and functional loss observed at birth (Heffez
devastating congenital defect. Children affected by MMC face et al., 1993; Meuli et al., 1995a; Meuli et al., 1995b). The denitive
signicant life-long physical disabilities including, leg paralysis secondary mechanisms underlying the pathogenesis of spinal
and sensory loss, bowel and bladder dysfunctions, skeletal cord injury remain unclear. Although, it has been proposed that
deformations, and the Arnold-Chiari II malformation with the in utero acquired damage of the spinal cord includes,
secondary hydrocephalus (Dias and McLone, 1993; Hunt, 1990; mechanical, degenerative, and possibly inammatory damage
Hunt and Poulton, 1995). These disabilities can lead to other to the openly exposed spinal cord tissue caused by exposure to
medical conditions that require clinical care. The most com- amniotic uid, traumatic injury within the amniotic space,
mon secondary conditions associated with MMC are urinary hydrodynamic pressure, or a combination of these (Heffez
tract infections followed by complications from devices/ et al., 1993; Meuli et al., 1995a; Meuli et al., 1995b; Meuli and
implants and various types of skin injuries e.g., pressure ulcers Moehrlen, 2013). Thus, prenatal therapeutic strategies that
resulting from prolonged sitting in one position (Dicianno and interrupt the pathological processes offer an appealing app-
Wilson, 2010; Verhoef et al., 2004). Approximately 3050% of roach for treatment of MMC.
MMC patients rely on a wheelchair for mobility (Hunt and
Oakeshott, 2003; McDonnell and McCann, 2000). The individuals 2.2. Prenatal and postnatal repair for MMC
living with the defect have a profoundly diminished quality of
life and ability to work. They usually require lifelong support Despite the clinical impact of MMC, there is no cure for this
and institutional care. To optimize the quality of life and devastating defect. Prevention strategies can reduce the onset of
improve the functional outcomes of these individuals, lifelong MMC; however, uncertainty remains regarding its etiology and
multidisciplinary care including neurosurgery, urology, ortho- the underlying mechanisms, which is a prerequisite for effective
pedics and rehabilitation is needed (Sandler, 2010). Treatment preventive strategies. Prophylaxis treatment with folic acid has
and management of patients with spina bida continue to have proven efcacious in reducing total numbers, however, it helps
a huge economic burden on the health care system (Centers for only a fraction of pregnant women, and thus a signicant
Disease Control and Prevention (CDC) 2007; Radcliff et al., 2012), portion of neural tube defects still persists (Oakley, 2010;
and its physical and emotional burden on children and their Osterhues et al., 2013; Parker et al., 2010). Once spina bida
families is devastating. occurs folic acid is not helpful. Therefore, women carrying a
fetus diagnosed with MMC have few options, including standard
2.1. Spinal cord injury in MMC surgical closure shortly after birth, closure of the defect during
the fetal period in selected cases, or termination of pregnancy.
MMC is a complex congenital defect of neural tube and vertebral Postnatal surgical closure of MMC defect is associated with poor
arches closure, characterized by a dynamic pathological process neurological outcomes because the postnatal therapy cannot
during gestation that results in the protrusion of the spinal cord prevent or reverse the neurological injury, since the affected
and meninges through a defect in the spine leading to the spinal spinal cord is destroyed and neurologic function lost before birth
cord injury at the level of MMC. The condition can result in (Adzick et al., 2011; Simpson and Greene, 2011). Because experi-
varying sensory and motor decits below the affected neurologic mental and clinical evidence shows progressive damage to the
level of the lesion, and a variety of associated problems, defective area during fetal development in utero, prenatal surgical
including hindbrain herniation, hydrocephaly, loss of bowel closure was thought to be a potential treatment (Heffez et al.,
and bladder control and various orthopedic complications (Dias 1990; Hutchins et al., 1996; Meuli et al., 1995b; Meuli et al., 1997;
and McLone, 1993; Hunt 1990; Hunt and Poulton, 1995; Hunt and Sival et al., 1997; Stiefel et al., 2007). Indeed, animal experime-
Oakeshott, 2003; Tomlinson and Sugarman, 1995). A better nts of in utero repair after surgically created defects showed
understanding of spinal cord injury in MMC has been revised improved neural function (Heffez et al., 1993; Meuli et al., 1996).
86 brain research 1619 (2015) 8490

Given that prenatal diagnosis of neural tube defects and surgical models of MMC created by fetal surgery provide evidence that
techniques have improved over the past several years, the open exposure of the normal spinal cord to the intrauterine
concept of in utero repair of MMC in humans became feasible. environment results in MMC-type lesion that have clinical
In utero surgical closure of MMC improves the functional neuro- and morphological similarities to human MMC (Heffez et al.,
logical outcomes including the ability to walk without orthotics 1990; Hutchins et al., 1996; Meuli et al., 1995b). Surgical
or devices in some patients, a reduction in hindbrain herniation lesions that remove the vertebral arches without directly
and a decreased need for ventriculoperitoneal shunting in damaging the spinal cord resulted in herniation of the spinal
patients who underwent prenatal compared with those who cord and its membranes through the vertebral defect, form-
had postnatal closure (Adzick et al., 2011; Simpson and Greene, ing a cystic human-like MMC (Meuli et al., 1995b). In addition,
2011). Currently, more than ten centers in the United States are earlier studies in primates also show that MMC created by
performing in utero MMC repair, which now is a clinical option fetal laminectomy resulted in the spinal cord injury and MMC-
for the management of prenatally diagnosed MMC in eligible like neurologic decits at birth (Michejda, 1984). Immediate in
patients (Moldenhauer, 2014). However, despite the supporting utero repair of the bone decit after fetal laminectomy resulted in
evidence that the prenatal surgical repair is more successful these animals having near normal neurological scores at birth
than postnatal MMC repair, the surgical procedure occur well (Michejda, 1984).
after the initial exposure of the spinal cord to the amniotic Although, several etiologies have been proposed for spina
environment, and does not completely repair the spinal cord bida, all with unknown casual mechanisms, it appears that the
injury, so signicant morbidity remains in most patients. In sentinel pathology in MMC is in the defect in the mesodermally-
addition, there are signicant maternal and fetal risks associated derived musculoskeletal tissues. The neurologic injury occurs
with prenatal surgery and the impact on the long-term out- secondary to these defects. Convincing experimental and clin-
comes of children who underwent fetal surgery for MMC is ical evidence indicates no signs of trauma or degeneration of
currently unknown and requires further study (Moldenhauer, spinal tissue at early gestational time points, and spinal cord
2014). Thus, further research of this defect is needed to advance degeneration appears to progress in severity with gestational
the understanding of MMC development and identify new age (Keller-Peck and Mullen, 1996; Meuli et al., 1997; Selcuki
treatment approaches, through which the concept of preventing et al., 2001; Stiefel et al., 2007). With naturally occurring MMC,
or reversing neurological impairments caused by MMC may be part of the spinal cord pushes through congenitally unclosed
achieved. openings in overlying vertebrae and protrudes from the fetus'
back into amniotic space, followed by progressive damage to the
2.3. Vertebral (mesenchymal) defects in MMC spinal cord leading in some cases to complete loss observed in
late stages of gestation (Hutchins et al., 1996; Meuli et al., 1997).
Spinal cord injury, which occurs with MMC is thought to be a The extent of neurological decits and disabilities associated
progressive intrauterine phenomenon, rather than comple- with MMC is dependent on the severity of the defect. Although,
tely a condition induced by defective neurulation. The etiol- the intrauterine conditions are thought to negatively impact
ogy and pathogenesis of MMC is unclear and most likely spinal cord development leading to functional loss observed at
mulifactorial. The accepted view has been that the neuroe- birth, there is little experimental data, e.g., from animal models
pithelium is initially involved in the failure of neural tube for the quantitative assessment of MMC pathology. Given the
closure and that the associated vertebral (mesenchymal) nature of the MMC defect, one potential quantitative means of
anomalies are secondary (Copp and Greene, 2013; Finnell assessing defect severity during gestation is by measuring
et al., 2000). While spina bida is often synonymous with impairment of fetal vertebral development with respect to the
neural tube defects, other etiologies have also been proposed degree of spinal cord damage, at different gestation periods. In
for spina bida development. Analysis of mouse models of combination with neurological assessment of animals, the
spina bida suggests that the mesoderm surrounding the information obtained from these studies would establish a
neural tube may be initially involved in development of this correlation between abnormal fetal spine development, the
defect (Helwig et al., 1995; Payne et al., 1997; Pickett et al., spinal cord injury and loss of neurologic function before birth.
2008; Takahashi et al., 1992). In experimental systems MMC- A comprehensive quantitative analysis of MMC pathology would
like defects can be induced by retinoic acid overexposure improve an assessment of MMC, while measurements at
(Alles and Sulik, 1990; Danzer et al., 2005; Tibbles and Wiley, different gestation periods should advance the understanding
1988). Although the exact mechanisms through which all- of the developmental trajectory of MMC.
trans retinoic acid induces MMC remains largely unknown, it
has been proposed that excessive cell death of embryonic 2.4. Quantitative assessment of MMC in an animal model
mesenchymal cells may play a role in the induction of these
malformations (Alles and Sulik, 1990). Early studies on Although several animal models of MMC have been developed
human fetuses with MMC suggest that the musculoskeletal to study the pathogenesis and treatment approaches, limited
system may be initially affected resulting in open exposure of work has been done to improve the quantitative outcome
the spinal cord, followed by progressive in utero damage to the measures of MMC. At present, anatomical MMC assessment
spinal cord (Hutchins et al., 1992; Jordan et al., 1991). These in animal models is usually performed by conventional 2D
ndings support the hypothesis that some categories of histopathological analysis of spinal cord defect and whole-body
human MMC may arise from a failure of the vertebrae to skeleton staining (Cai et al., 2007; Danzer et al., 2005). While
close at the midline, suggesting that the primary abnormality histological methods offer unique capabilities, these trad-
may be in the musculoskeletal system. Accordingly, animal itional techniques may be inadequate to fully visualize and
brain research 1619 (2015) 8490 87

quantitatively evaluate MMC defects. The methods of whole- reported in the literature, micro-CT is the imaging technique of
body skeleton staining are invasive, destructive techniques that choice for the measurement and visualization of bone structure.
severely limit precise 3D visualization and quantitative assess- It can also be used for quantitative analysis of various fetal
ment of skeletal defects in MMC fetuses and are time consum- skeletal development parameters including bone mineral den-
ing. The invasiveness and 2D analyses of histological sections sity as well as identication of skeletal malformations with
are also the main disadvantages limiting the opportunity for high degree of accuracy (Campbell and Sophocleous, 2014;
full 3D visualization and quantitative evaluation of fetal spinal Cunningham and Black, 2009; Oest et al., 2008). Micro CT has
cord abnormalities in MMC. In addition, the complete assess- also become a popular imaging technique to study soft tissues
ment of MMC defects can be difcult to perform in the same (Campbell and Sophocleous, 2014). Recent, studies indicate that
animal using traditional techniques. For example, due to the ex vivo micro-CT imaging, with the aid of staining agents that
destructive nature of histological techniques, analysis of the make soft tissues radiopaque, has the potential for non-invasive
vertebral level and skin level of the MMC lesion in mice after imaging and quantitative analysis of soft tissues in 3D including
whole skeletal preparations was performed using the correla- spinal cord (Saito and Murase, 2013; Tahara and Larsson, 2013;
tion of the cranial end of the skeletal defect with the suture- Vegesna et al., 2012; Vegesna et al., 2013). Although, the opti-
marked cranial end of the skin defect (Stiefel and Meuli, 2008). mization of staining methods is needed to distinguish soft tissue
Advanced techniques, e.g. MRI can provide 3D images of MMC from bone tissue and delineate spinal cord, these studies
in animal model (Danzer et al., 2005) however, data acquisition indicate the potential of micro-CT for evaluation of both verteb-
is usually time-consuming and it is difcult to obtain high- ral and spinal cord defects within the same animal. The major
resolution imaging in small animals adequate for quantitative advantage of high resolution micro-CT is the ability to provide
analysis of MMC, in particular the skeletal defect. detailed images of the anatomy that can be visualized in both 2D
Micro-computed tomography (micro-CT) is a non-invasive and 3D and enables quantitative digital analyses. Micro-CT is a
whole volume imaging technique that provides high contrast highly versatile imaging method, and with appropriate contrast
between soft tissue and bone and offers quantitative data on stains can produce high-quality images of embryo at wide range
bone morphology and architecture and has been helpful in of developmental stages (Metscher, 2011), which makes micro-
characterizing developmental defects of bone (Bouxsein et al., CT an attractive technique for analysis of MMC defects at early
2010; Ford-Hutchinson et al., 2003; Oest et al., 2008; Vasquez embryonic stages. Morphometric analyses at different gestation
et al., 2013). The great detail with which the anatomy can be periods can provide better characteristics of the severity of bone
visualized and the abnormalities quantied (Cunningham and and spinal cord damage in MMC during lesion progression. This
Black, 2009; Oest et al., 2008), makes high resolution micro-CT an high resolution micro-CT technique would also be relevant for
attractive imaging technique for assessing the characteristics of quantitative measurements of MMC repair by combining anato-
MMC skeletal defects in animal models. As an initial step toward mical data with improvement in neonatal neurologic function,
the development of improved methods for assessment of MMC, which presently is difcult to study even in experimental
we have recently described the assessment of MMC skeletal models.
defects in an established retinoic acid induced rat model of
lumbosacral MMC using high-resolution micro-CT imaging com-
bined with digital quantication methods (Barbe et al., 2014). The 3. Future directions
retinoic acid induced rat model of MMC is developmentally and
phenotypically analogous to human MMC, and is relevant for Spina bida is one of the more common congenital malfor-
investigating the pathogenesis and in utero treatments of MMC mations in humans, for which MMC is the most severe form
(Danzer et al., 2005; Danzer et al., 2011; Watanabe et al., 2011; associated with signicant long-term complications (Meuli
Zhao et al., 2008). Using 3D micro-CT images of rat vertebral and Moehrlen, 2013; Parker et al., 2010). Despite the clinical
defects and corresponding normal vertebra we observed, in impact of MMC, there is no effective treatment for MMC.
detail, skeletal defects in lumbosacral vertebra of MMC rats, Historically, the primary treatment for MMC, and other spinal
including morphological defects of individual dorsal vertebral cord disorders, was palliative, treating the symptoms and
arches. The defect in lambosacral vertebra appeared in the form provide supportive care rather than to cure and/or modify the
of delayed or absent ossication. Buttery-shaped, dumbbell- disease state. Currently, surgical repair of MMC before or after
shaped, non-fused, and absent to nearly absent vertebra centra birth is considered to be a treatment option for some cases
were identied using high resolution 3D micro-CT images of but still suffers from signicant limitations in repairing MMC
lumbosacral vertebrae (Barbe et al., 2014). We show high- defects. However, recent research on stem cells and tissue
resolution micro-CT to be an especially powerful method of engineering warrants the possibility of actually reversing the
analysis that provides not only anatomical details of MMC course of some neurologic disorders by repairing, replacing
defect, but also adequate 2D and 3D images for digital quanti- and/or regeneration of damaged cells (Thwaites et al., 2012;
cation not previously achieved in models of MMC. We were Wong et al., 2014). Tissue engineering holds great promise for
able to nondestructively quantify the distance between the ends the future treatment of birth defects present in MMC;
of the vertebral arches in the retinoic acid induced rat model of because, the ultimate pathology in these conditions is lack
MMC and showed a signicant increase in the distance between of proper development or loss of tissue. Accordingly, a
dorsal vertebral arches from L5 to S4 when compared to the modest but growing body of research indicates that prenatal
control group. In addition, micro-CT method is a powerful tool repair of MMC by tissue engineering approaches may be
for assessing overall skeletal phenotype and it allowed us to promising; however, thus far the documentation of resu-
visualize supernumerary ribs in fetuses with MMC. As often lts is very limited and it lacks important information on
88 brain research 1619 (2015) 8490

evaluation of newly formed tissues, making interpretation quantitative assessment of animal models of MMC and inter-
difcult. For example not all studies performed histological pretation of results because destructive methods of whole-body
analyses of repaired MMC and some only described histolo- skeleton staining and 2D analysis of spinal cord provide only
gical results without the photodocumentation to support the discrete information and often incomplete representation of 3D
claim (for review see (Watanabe et al., 2014)). This may stem environment.
from the fact that the assessment of new tissue formation Besides its impact on the advance of testing prenatal treat-
using traditional histological techniques can be time con- ments for MMC, the use of advanced methods such as micro-CT
suming and difcult in fetuses with MMC. One of the most for a 3D visualization and quantitative assessment of bone and
promising prenatal approaches for repair of MMC is the use of soft tissue damage in MMC should aid in more accurate des-
scaffolds in combination with stem cells and/or signaling criptions of MMC pathology in studies on the progression of
molecules for 3D tissue formation. Efcient methodologies MMC during gestation in animal models. Although the micro-
that provide full 3D visualization and quantitative assess- CT-system is not capable of molecular or cellular assessments as
ment of newly formed tissues are thus necessary in order to traditional methods are, but as needed, this limitation can be
compare the efcacy of different treatment strategies as well circumvented by the use of immunohistochemistry in conjunc-
as establish a correlation between the anatomical repair and tion with micro-CT methods (Iwasaki et al., 2012).
neurological functional outcomes in studies of MMC repair. In In summary, it is evident that further improvements in
light of the above considerations, it appears that the use of prenatal and preventive MMC treatment approaches are needed
high resolution 3D micro-CT imaging with and without to advance treatments of MMC. However, a deeper understand-
contrast agents that has a potential to visualize the MMC ing of MMC pathogenesis is a prerequisite for effective prevent-
anatomy with a micron resolution and enables quantitative ive and therapeutic strategies. We propose that the inclusion of
digital analyses of MMC, may serve as a non-destructive 3D imaging techniques such as micro-CT for visualization and
alternative, or adjunct to histological methods for 3D visua- quantitative measures of musculoskeletal and spinal cord def-
lization and/or quantication of MMC repair. ects in animal models of MMC will aid in a deeper understanding
Even though the prenatal treatment of MMC is improving of MMC pathogenesis and provide quantitative and 3D informa-
with better understanding of the pathophysiology associated tion on repair of MMC that are essential to determine the efcacy
with MMC (Meuli and Moehrlen, 2013; Moldenhauer, 2014; of treatment strategies in preclinical animal models. Finally,
Watanabe et al., 2014), it is evident that further improve- standardized and quantitative outcome measures are needed to
ments in prenatal treatment approaches and deeper under- compare the efcacy of different treatment strategies and
standing of MMC pathogenesis are needed. Currently, the improve the efciency of MMC studies.
research for prenatal treatment of MMC mainly focuses on
approaches for providing soft tissue coverage of the MMC
defect in gestation to prevent amniotic-uid induced spinal
cord damage and/or attempts for achieving neuroprotection Acknowledgments
and potentially spinal cord regeneration (Watanabe et al.,
2014). However, it is likely that in the future the preventive The authors thank Dr. Mary F. Barbe and colleagues from the
strategies and the therapeutic combinations affecting also Department of Anatomy and Cell Biology, Temple University
the vertebral defect will be necessary to substantially School of Medicine for providing micro-computed tomography
improve the MMC treatment. Owing to the important role of equipment and expertise, which was essential to the evaluation
micro-CT in studies of bone repair and quantitative assess- of MMC in a rat model. The authors also thank members of
ment of 3D tissue formation (Campbell and Sophocleous, SHPRC for their support and sharing of ideas. Grant support from
2014; Guldberg et al., 2008), the inclusion of 3D micro-CT Shriners Hospitals (85230-PHI and 86400-PHI) is gratefully
imaging to complement histological evaluation of MMC repair acknowledged.
would be essential to determine the treatment efcacy of
different treatment strategies in future studies. r e f e r e n c e s
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